Optimized expression of HPV31 L1 in yeast

ABSTRACT

Synthetic DNA molecules encoding the HPV31 L1 protein are provided. Specifically, the present invention provides polynucleotides encoding HPV31 L1 protein, wherein said polynucleotides are free from internal transcription termination signals that are recognized by yeast. Also provided are synthetic polynucleotides encoding HPV31 L1 wherein the polynucleotides have been codon-optimized for high level expression in a yeast cell. The synthetic molecules may be used to produce HPV31 virus-like particles (VLPs), and to produce vaccines and pharmaceutical compositions comprising the HPV31 VLPs. The vaccines of the present invention provide effective immunoprophylaxis against papillomavirus infection through neutralizing antibody and cell-mediated immunity 
     
       
         
               
             
                 HPV31 L1 total rebuild nucleotide and amino 
               
                 acid sequence: 
               
                   
               
                      M  S  L  W   R  P  S 
               
                    1 ATGTCTTTGT GGAGACCATC 
               
                   
               
               
               
             
                   
                  E  A  T   V  Y  L  P   P  V  P 
               
                   
                 TGAAGCTACC GTCTACTTGC CACCAGTCCC 
               
                   
                   
               
               
             
                       V  S  K   V  V  S  T 
               
                   51 AGTCTCTAAG GTCGTCTCTA 
               
                   
               
               
               
             
                   
                   D  E  Y   V  T  R   T  N  I  Y 
               
                   
                 CCGACGAATA CGTCACCAGA ACCAACATCT 
               
                   
                   
               
               
             
                        Y  H  A   G  S  A 
               
                  101 ACTACCACGC TGGTTCTGCT 
               
                   
               
               
               
             
                   
                 R  L  L  T   V  G  H   P  Y  Y 
               
                   
                 AGATTGTTGA CCGTCGGTCA CCCATACTAC 
               
                   
                   
               
               
             
                      S  I  P  K   S  D  N 
               
                  151 TCTATCCCAA AGTCTGACAA 
               
                   
               
               
               
             
                   
                  P  K  K   I  V  V  P   K  V  S 
               
                   
                 CCCAAAGAAG ATCGTCGTCC CAAAGGTCTC 
               
                   
                   
               
               
             
                       G  L  Q   Y  R  V  F 
               
                  201 TGGTTTGCAA TACAGAGTCT 
               
                   
               
               
               
             
                   
                   R  V  R   L  P  D   P  N  K  F 
               
                   
                 TCAGAGTCAG ATTGCCAGAC CCAAACAAGT 
               
                   
                   
               
               
             
                        G  F  P   D  T  S 
               
                  251 TCGGTTTCCC AGACACCTCT 
               
                   
               
               
               
             
                   
                 F  Y  N  P   E  T  Q   R  L  V 
               
                   
                 TTCTACAACC CAGAAACCCA AAGATTGGTC 
               
                   
                   
               
               
             
                      W  A  C  V   G  L  E 
               
                  301 TGGGCTTGTG TCGGTTTGGA 
               
                   
               
               
               
             
                   
                  V  G  R   G  Q  P  L   G  V  G 
               
                   
                 AGTCGGTAGA GGTCAACCAT TGGGTGTCGG 
               
                   
                   
               
               
             
                       I  S  G   H  P  L  L 
               
                  351 TATCTCTGGT CACCCATTGT 
               
                   
               
               
               
             
                   
                   N  K  F   D  D  T   E  N  S  N 
               
                   
                 TGAACAAGTT CGACGACACC GAAAACTCTA 
               
                   
                   
               
               
             
                        R  Y  A   G  G  P 
               
                  401 ACAGATACGC TGGTGGTCCA 
               
                   
               
               
               
             
                   
                 G  T  D  N   R  E  C   I  S  M 
               
                   
                 GGTACCGACA ACAGAGAATG TATCTCTATG 
               
                   
                   
               
               
             
                      D  Y  K  Q   T  Q  L 
               
                  451 GACTACAAGC AAACCCAATT 
               
                   
               
               
               
             
                   
                  C  L  L   G  C  K  P   P  I  G 
               
                   
                 GTGTTTGTTG GGTTGTAAGC CACCAATCGG 
               
                   
                   
               
               
             
                       E  H  W   G  K  G  S 
               
                  501 TGAACACTGG GGTAAGGGTT 
               
                   
               
               
               
             
                   
                   P  C  S   N  N  A   I  T  P  G 
               
                   
                 CTCCATGTTC TAACAACGCT ATCACCCCAG 
               
                   
                   
               
               
             
                        D  C  P    P L  E 
               
                  551 GTGACTGTCC ACCATTGGAA 
               
                   
               
               
               
             
                   
                 L  K  N  S   V  I  Q   D  G  D 
               
                   
                 TTGAAGAACT CTGTCATCCA AGACGGTGAC 
               
                   
                   
               
               
             
                      N  V  D  T   G  F  G 
               
                  601 ATGGTCGACA CCGGTTTCGG 
               
                   
               
               
               
             
                   
                  A  N  D   F  T  A  L   Q  D  T 
               
                   
                 TGCTATGGAC TTCACCGCTT TGCAAGACAC 
               
                   
                   
               
               
             
                       K  S  W   V  P  L  D 
               
                  651 CAAGTCTAAC GTCCCATTGG 
               
                   
               
               
               
             
                   
                   I  C  N   S  I  C   K  Y  P  D 
               
                   
                 ACATCTGTAA CTCTATCTGT AAGTACCCAG 
               
                   
                   
               
               
             
                        Y  L  K   M  V  A 
               
                  701 ACTACTTGAA GATGGTCGCT 
               
                   
               
               
               
             
                   
                 E  P  Y  G   D  T  L   F  F  Y 
               
                   
                 GAACCATACG GCGACACCTT GTTCTTCTAC 
               
                   
                   
               
               
             
                       L R  R  E   Q  M  F 
               
                  751 TTGCGTAGAG AACAGATGTT 
               
                   
               
               
               
             
                   
                  V  R  H   F  F  N  R   S  G  T 
               
                   
                 CGTAAGGCAC TTCTTCAACA GATCCGGCAC 
               
                   
                   
               
               
             
                       V  G  E   S  V  P  T 
               
                  801 CGTAGGTGAA TCTGTCCCAA 
               
                   
               
               
               
             
                   
                   D  L  Y   I  K  G   S  G  S  T 
               
                   
                 CCGACCTGTA CATCAAGGGC TCCGGTTCCA 
               
                   
                   
               
               
             
                        A  T  L   A  N  S 
               
                  851 CCGCTACCCT GGCTAACTCC 
               
                   
               
               
               
             
                   
                 T  Y  F  P   T  P  S   G  S  N 
               
                   
                 ACCTACTTCC CAACTCCATC TGGCTCCATG 
               
                   
                   
               
               
             
                      V  T  S  D   A  Q  I 
               
                  901 GTCACCTCCG ACGCTCAGAT 
               
                   
               
               
               
             
                   
                  F  N  K   P  Y  W  M   Q  R  A 
               
                   
                 CTTCAACAAG CCATACTGGA TGCAGCGTGC 
               
                   
                   
               
               
             
                       Q  G  H   N  N  G  I 
               
                  951 ACAGGGTCAC AACAACGGTA 
               
                   
               
               
               
             
                   
                   C  W  G   N  Q  L   F  V  T  V 
               
                   
                 TCTGTTGGGG TAACCAGCTG TTCGTGACTG 
               
                   
                   
               
               
             
                        V  D  T   T  R  S 
               
                 1001 TGGTCGATAC CACGCGTTCT 
               
                   
               
               
               
             
                   
                 T  N  N  S   V  C  A  A  I   A 
               
                   
                 ACCAACATGT CTGTCTGTGC TGCAATCGCT 
               
                   
                   
               
               
             
                      N  S  D  T   T  F  K 
               
                 1051 AACTCTGACA CTACCTTCAA 
               
                   
               
               
               
             
                   
                  S  S  N   F  K  E  Y   L  R  H 
               
                   
                 GTCCTCTAAC TTCAAGGAGT ACCTGAGACA 
               
                   
                   
               
               
             
                       G  E  E   F  D  L  Q 
               
                 1101 TGGTGAGGAA TTCGATCTGC 
               
                   
               
               
               
             
                   
                   F  I  F   Q  L  C   K  I  T  L 
               
                   
                 AATTCATCTT CCAGTTGTGC AAGATCACCC 
               
                   
                   
               
               
             
                        S  A  D   I  N  T 
               
                 1151 TGTCTGCTGA CATCATGACC 
               
                   
               
               
               
             
                   
                 Y  I  H  S   M  N  P   A  I  L 
               
                   
                 TACATCCACA GTATGAACCC TGCCATCCTG 
               
                   
                   
               
               
             
                      E  D  W  N   F  G  L 
               
                 1201 GAGGACTGGA ACTTCGGTCT 
               
                   
               
               
               
             
                   
                  T  T  P   P  S  G  S   L  E  D 
               
                   
                 GACCACTCCA CCTTCCGGTT CTTTGGAAGA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US04/08677, international filing dateof Mar. 19, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/457,172 filed Mar. 24, 2003, the contents of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the therapy of humanpapillomavirus (HPV). More specifically, the present invention relatesto synthetic polynucleotides encoding HPV31 L1 protein, and torecombinant vectors and hosts comprising said polynucleotides. Thisinvention also relates to HPV31 virus-like particles (VLPs) and to theiruse in vaccines and pharmaceutical compositions for preventing andtreating HPV.

BACKGROUND OF THE INVENTION

There are more than 80 types of human papillomavirus (HPV), many ofwhich have been associated with a wide variety of biological phenotypes,from benign proliferative warts to malignant carcinomas (for review, seeMcMurray et al., Int. J. Exp. Pathol. 82(1): 15-33 (2001)). HPV6 andHPV11 are the types most commonly associated with benign warts,nonmalignant condylomata acuminate and/or low-grade dysplasia of thegenital or respiratory mucosa. HPV16 and HPV18 are the high-risk typesmost frequently associated with in situ and invasive carcinomas of thecervix, vagina, vulva and anal canal. More than 90% of cervicalcarcinomas are associated with infections of HPV16, HPV18 or the lessprevalent oncogenic types HPV31, -33, -45, -52 and -58 (Schiffman etal., J. Natl. Cancer Inst. 85(12): 958-64 (1993)). The observation thatHPV DNA is detected in 90-100% of cervical cancers provides strongepidemiological evidence that HPVs cause cervical carcinoma (see Boschet al., J. Clin. Pathol. 55: 244-265 (2002)).

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode up to eight early and two late genes. The openreading frames (ORFs) of the viral genomes are designated E1 to E7, andL1 and L2, where “E” denotes early and “L” denotes late. L1 and L2 viruscapsid proteins, while the E genes are associated with functions such asviral replication and cellular transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. The L2 protein is a minor capsid protein. Immunological datasuggest that most of the L2 protein is internal to the L1 protein. Boththe L1 and L2 proteins are highly conserved among differentpapillomaviruses.

Expression of the L1 protein or a combination of the L1 and L2 proteinsin yeast, insect cells, mammalian cells or bacteria leads toself-assembly of virus-like particles (VLPs) (for review, see Schillerand Roden, in Papillomavirus Reviews: Current Research onPapillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, pp101-12 (1996)). VLPs are morphologically similar to authentic virionsand are capable of inducing high titers of neutralizing antibodies uponadministration into an animal or a human. Because VLPs do not containthe potentially oncogenic viral genome, they present a safe alternativeto use of live virus in HPV vaccine development (for review, seeSchiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000)). For thisreason, the L1 and L2 genes have been identified as immunologicaltargets for the development of prophylactic and therapeutic vaccines forHPV infection and disease.

HPV vaccine development and commercialization have been hindered bydifficulties associated with obtaining high expression levels of capsidproteins in successfully transformed host organisms, limiting theproduction of purified protein. Therefore, despite the identification ofwild-type nucleotide sequences encoding HPV L1 proteins such as HPV31 L1proteins (Goldsborough et al., Virology 171(1): 306-311 (1989), it wouldbe highly desirable to develop a readily renewable source of crude HPVproteins that utilizes HPV31 L1-encoding nucleotide sequences that areoptimized for expression in the intended host cell. Additionally, itwould be useful to produce large quantities of HPV31 L1 VLPs having theimmunity-conferring properties of the native proteins for use in vaccinedevelopment.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods to elicit orenhance immunity to the protein products expressed by HPV31 L1 genes,which have been associated with cervical cancer. Specifically, thepresent invention provides polynucleotides encoding HPV31 L1 protein,wherein said polynucleotides are free from internal transcriptiontermination signals that are recognized by yeast. Also provided aresynthetic polynucleotides encoding HPV31 L1 wherein the polynucleotideshave been codon-optimized for high level expression in a yeast cell. Thepresent invention further provides HPV31 virus-like particles (VLPs) anddiscloses use of said VLPs in immunogenic compositions and vaccines forthe prevention and/or treatment of HPV disease or HPV-associated cancer.

The present invention relates to synthetic DNA molecules encoding theHPV31 L1 protein. In one aspect of the invention, the nucleotidesequence of the synthetic molecule is altered to eliminate transcriptiontermination signals that are recognized by yeast. In another aspect, thecodons of the synthetic molecules are designed so as to use the codonspreferred by a yeast cell. The synthetic molecules may be used as asource of HPV31 L1 protein, which may self-assemble into VLPs. Said VLPsmay be used in a VLP-based vaccine.

A particular embodiment of the present invention comprises a syntheticnucleic acid molecule which encodes the HPV31 L1 protein as set forth inSEQ ID NO:4, said nucleic acid molecule comprising a sequence ofnucleotides as set forth in SEQ ID NO:2 or SEQ ID NO:3.

As stated above, provided herein are synthetic polynucleotides encodingthe HPV31 L1 gene which are free from transcription termination signalsthat are recognized by yeast. This invention also provides syntheticpolynucleotides encoding HPV 31 L1 as described, which are furtheraltered so as to contain codons that are preferred by yeast cells.

Also provided are recombinant vectors and recombinant host cells, bothprokaryotic and eukaryotic, which contain the nucleic acid moleculesdisclosed throughout this specification.

The present invention relates to a process for expressing an HPV31 L1protein in a recombinant host cell, comprising: (a) introducing a vectorcomprising a nucleic acid encoding an HPV31 L1 protein into a yeast hostcell; wherein the nucleic acid molecule is free from internaltranscription termination signals that are recognized by yeast and; (b)culturing the yeast host cell under conditions which allow expression ofsaid HPV31 L1 protein.

The present invention further relates to a process for expressing anHPV31 L1 protein in a recombinant host cell, comprising: (a) introducinga vector comprising a nucleic acid encoding an HPV31 L1 protein into ayeast host cell; wherein the nucleic acid molecule is codon-optimizedfor optimal expression in the yeast host cell and; (b) culturing theyeast host cell under conditions which allow expression of said HPV31 L1protein.

In preferred embodiments, the nucleic acid comprises a sequence ofnucleotides as set forth in SEQ ID NO:2 or SEQ ID NO:3.

This invention also relates to HPV31 virus-like particles (VLPs),methods of producing HPV31 VLPs, and methods of using HPV31 VLPs.

In a preferred embodiment of the invention, the HPV31 VLPs are producedin yeast. In a further preferred embodiment, the yeast is selected fromthe group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha,Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, andSchizosaccharomyces pombe.

Another aspect of this invention is an HPV31 VLP, which comprises anHPV31 L1 protein produced by a HPV31 L1 gene which is free fromtranscription termination signals that are recognized by yeast.

Yet another aspect of this invention is an HPV31 VLP, which comprises anHPV31 L1 protein produced by a codon-optimized HPV31 L1 gene. In apreferred embodiment of this aspect of the invention, thecodon-optimized HPV31 L1 gene consists essentially of a sequence ofnucleotides as set forth in SEQ ID NO:2 or SEQ ID NO:3.

This invention also provides a method for inducing an immune response inan animal comprising administering HPV31 virus-like particles to theanimal. In a preferred embodiment, the HPV31 VLPs are produced by acodon-optimized gene. In a further preferred embodiment, the HPV31 VLPsare produced by a gene that is free from transcription terminationsequences that are recognized by yeast.

Yet another aspect of this invention is a method of preventing ortreating HPV-associated cervical cancer comprising administering to amammal a vaccine comprising HPV31 VLPs. In a preferred embodiment ofthis aspect of the invention, the HPV31 VLPs are produced in yeast.

This invention also relates to a vaccine comprising HPV31 virus-likeparticles (VLPs).

In an alternative embodiment of this aspect of the invention, thevaccine further comprises VLPs of at least one additional HPV type. In apreferred embodiment, the at least one additional HPV type is selectedfrom the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35,HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59, and HPV68.

This invention also relates to pharmaceutical compositions comprisingHPV 31 virus-like particles. Further, this invention relates topharmaceutical compositions comprising HPV31 VLPs and VLPs of at leastone additional HPV type. In a preferred embodiment, the at least oneadditional HPV type is selected from the group consisting of: HPV6,HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55,HPV56, HPV58, HPV59, and HPV68.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

As used throughout the specification and appended claims, the followingdefinitions and abbreviations apply:

The term “promoter” refers to a recognition site on a DNA strand towhich the RNA polymerase binds. The promoter forms an initiation complexwith RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences termed “enhancers” or“upstream activating sequences” or inhibiting sequences termed“silencers”.

The term “vector” refers to some means by which DNA fragments can beintroduced into a host organism or host tissue. There are various typesof vectors including plasmid, virus (including adenovirus),bacteriophages and cosmids.

The designation “31 L1 wild-type sequence” refers to the HPV31 L1sequence disclosed herein as SEQ ID NO:1. Although the HPV 31 L1wild-type sequence has been described previously, it is not uncommon tofind minor sequence variations between DNAs obtained from clinicalisolates. Therefore, a representative HPV31 L1 wild-type sequence wasisolated from clinical samples previously shown to contain HPV 31 DNA(see EXAMPLE 1). The 31 L1 wild-type sequence was used as a referencesequence to compare the codon-optimized HPV 31 L1 sequences disclosedherein (see FIG. 1).

The designation “31 L1 partial rebuild” refers to a construct, disclosedherein (SEQ ID NO:2), in which the HPV31 L1 nucleotide sequence waspartially rebuilt to contain yeast-preferred codons for optimalexpression in yeast. The 31 L1 partial rebuild comprises alterations inthe middle portion of the HPV 31 L1 wild-type nucleotide sequence(nucleotides 697-1249). The complete HPV 31 L1 sequence was also rebuiltwith yeast-preferred codons, which is referred to herein as the “31 L1total rebuild” (SEQ ID NO:3).

The term “effective amount” means sufficient vaccine composition isintroduced to produce the adequate levels of the polypeptide, so that animmune response results. One skilled in the art recognizes that thislevel may vary.

A “conservative amino acid substitution” refers to the replacement ofone amino acid residue by another, chemically similar, amino acidresidue. Examples of such conservative substitutions are: substitutionof one hydrophobic residue (isoleucine, leucine, valine, or methionine)for another; substitution of one polar residue for another polar residueof the same charge (e.g., arginine for lysine; glutamic acid foraspartic acid).

The term “mammalian” refers to any mammal, including a human being.

“VLP” or “VLPs” mean(s) virus-like particle or virus-like particles.

“Synthetic” means that the HPV31 L1 gene has been modified so that itcontains a sequence of nucleotides that is not the same as the sequenceof nucleotides present in the naturally occurring wild-type HPV31 L1gene. As stated above, synthetic molecules are provided hereincomprising a sequence of nucleotides that are altered to eliminatetranscription termination signals recognized by yeast. Also providedherein are synthetic molecules comprising codons that are preferred forexpression by yeast cells. The synthetic molecules provided hereinencode the same amino acid sequences as the wild-type HPV31 L1 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment showing nucleotides that were altered inthe partial (SEQ ID NO:2) and total rebuild (SEQ ID NO:3) 31 L1 genes(See EXAMPLE 2). The reference sequence is the 31 L1 wild-type sequence(SEQ ID NO:1; see EXAMPLE 1). Nucleotides in the 31 L1 partial and totalrebuild sequences that are identical to the reference sequence areindicated with dots. Altered nucleotides are indicated at theircorresponding location. Nucleotide number is contained within theparentheses.

FIG. 2 shows the 31 L1 total rebuild nucleotide (SEQ ID NO:3) and aminoacid sequences (SEQ ID NO:4). The nucleotide number is indicated on theleft.

FIG. 3 summarizes the changes between the three HPV 31 L1 sequenceconstructs, which are listed on the left. The fourth column indicatesthe percent nucleotide identity between the indicated construct and the31 L1 wild-type sequence and the fifth column indicates the amino acididentity. The last column indicates the number of nucleotides that werealtered to yeast-preferred codon sequences and the region where thealterations were made.

FIG. 4 shows a Northern blot probed specifically for HPV 31 L1 underhigh stringency (see EXAMPLE 4). Arrows on the left indicate theposition of the HPV 31 L1 full length and truncated transcripts. Laneslabeled “31 wt” are from the same RNA preparation of yeast containing 31L1 wild-type sequences. The lane labeled “16” contains RNA from HPV16,which is not recognized by the HPV 31 L1 probe because of the highstringency conditions. The lane labeled “Neg” is a yeast extractcontaining no L1 coding sequences. Lanes labeled “31 R” are from RNA oftwo separate isolated colonies expressing the 31 L1 partial-rebuildsequence.

FIG. 5 shows a portion of the data from two capture radioimmunoassay(RIA) experiments in counts per minute (cpm)/mg total protein (seeEXAMPLE 7). Cpm obtained in the RIA are a relative indicator of HPV 31L1 VLPs. The RIA data demonstrate increased 31 L1 VLP expression inyeast protein extracts from yeast-preferred codon rebuilt genesequences.

FIG. 6 shows a representative sample of the 31 L1 VLPs described herein,as visualized by transmission electron microscopy (see EXAMPLE 8). Thebar represents 100 nm.

DETAILED DESCRIPTION OF THE INVENTION

The majority of cervical carcinomas are associated with infections ofspecific oncogenic types of human papillomavirus (HPV). The presentinvention relates to compositions and methods to elicit or enhanceimmunity to the protein products expressed by genes of oncogenic HPVtypes. Specifically, the present invention provides polynucleotidesencoding HPV31 L1 and HPV31 virus-like particles (VLPs) and disclosesuse of said polynucleotides and VLPs in immunogenic compositions andvaccines for the prevention and/or treatment of HPV-associated cancer.

The wild-type HPV31 L1 nucleotide sequence has been reported(Goldsborough et al., Virology 171(1): 306-311 (1989); Genbank Accession# J04353). The present invention provides synthetic DNA moleculesencoding the HPV31 L1 protein. The synthetic molecules of the presentinvention comprise a sequence of nucleotides, wherein some of thenucleotides have been altered so as to eliminate transcriptiontermination signals that are recognized by yeast. In alternativeembodiments, the codons of the synthetic molecules are designed so as touse the codons preferred by a yeast cell for high-level expression. Thesynthetic molecules may be used as a source of HPV31 L1 protein, whichmay self-assemble into VLPs. Said VLPs may be used in a VLP-basedvaccine to provide effective immunoprophylaxis against papillomavirusinfection through neutralizing antibody and cell-mediated immunity. SuchVLP-based vaccines are also useful for treatment of already establishedHPV infections.

Expression of HPV VLPs in yeast cells offers the advantages of beingcost-effective and easily adapted to large-scale growth in fermenters.However, many HPV L1 proteins, including HPV31 L1 (see EXAMPLE 4), areexpressed at low levels in yeast cells. It has been determined inaccordance with the present invention that low level expression of HPV31L1 is due to truncation of the mRNA transcript resulting from thepresence of transcription termination signals that are recognized byyeast. By altering the HPV31 L1 DNA to eliminate any potential sequencesresembling yeast transcription termination sites, it is possible tofacilitate the transcription of full-length mRNA resulting in increasedHPV31 L1 protein expression.

Accordingly, in some embodiments of this invention, alterations havebeen made to the HPV31 L1 DNA to eliminate any potential sequencesresembling yeast transcription termination signals. These alterationsallow expression of the full-length HPV31 transcript, as opposed to atruncated transcript (see EXAMPLE 4), improving expression yield.

As noted above, synthetic DNAs of the present invention comprisealterations from the wild-type HPV31 L1 sequence that were made toeliminate yeast-recognized transcription termination sites. One of skillin the art will recognize that additional DNA molecules can beconstructed that encode the HPV31 L1 protein, but do not contain yeasttranscription termination sites. Techniques for finding yeasttranscription termination sequences are well known in the art.Transcription termination and 3′ end formation of yeast mRNAs requiresthe presence of three signals: (1) an efficiency element such as TATATAor related sequences, which enhances the efficiency of positioningelements located downstream; (2) positioning element(s), which determinethe location of the poly(A) site and (3) the polyadenylation site(usually Py(A)n).

The scientific literature is replete with descriptions of sequences thatencode yeast transcription termination signals. See, for example, Guoand Sherman, Trends Biochem. Sci. 21: 477-481 (1986); Guo and Sherman,Mol. Cell. Biol. 16(6): 2772-2776 (1996); Zaret et al, Cell 28:563-573(1982); Henikoff et al, Cell 33:607-614 (1983); Thalenfeld et al, J.Biol. Chem. 258(23):14065-14068 (1983); Zaret et al, J. Mol. Biol.176:107-135 (1984); Heidmann et al, Mol. Cell Biol 14:4633-4642 (1984);and Russo, Yeast 11:447-453 (1985). Therefore, one of skill in the artwould have no difficulty determining which sequences to avoid in orderto construct a synthetic HPV31 L1 gene that produces a full-length mRNAtranscript in accordance with the present invention. Additionally,assays and procedures to assess whether a yeast transcriptiontermination sequence is present within the synthetic sequence are wellestablished in the art, so that an ordinary skilled artisan would beable to determine if a constructed HPV31 L1 sequence comprisestermination sequences that need to be eliminated.

As described above, the present invention relates to a nucleic acidmolecule encoding HPV type 31 L1 protein, the nucleic acid moleculebeing free from internal transcription termination signals which arerecognized by yeast. In exemplary embodiments of the invention, thesynthetic nucleic acid molecules comprise a sequence of nucleotides asset forth in SEQ ID NO:2 or SEQ ID NO:3.

In alternative embodiments of the present invention, HPV31 L1 genesequences are “optimized” for high level expression in a yeast cellularenvironment. Codon-optimized HPV31 L1 genes contemplated by the presentinvention include synthetic molecules encoding HPV31 L1 that are freefrom internal transcription termination signals which are recognized byyeast, further comprising at least one codon that is codon-optimized forhigh level expression in yeast cells.

A “triplet” codon of four possible nucleotide bases can exist in over 60variant forms. Because these codons provide the message for only 20different amino acids (as well as transcription initiation andtermination), some amino acids can be coded for by more than one codon,a phenomenon known as codon redundancy. For reasons not completelyunderstood, alternative codons are not uniformly present in theendogenous DNA of differing types of cells. Indeed, there appears toexist a variable natural hierarchy or “preference” for certain codons incertain types of cells. As one example, the amino acid leucine isspecified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA,and TTG. Exhaustive analysis of genome codon frequencies formicroorganisms has revealed endogenous DNA of E. coli most commonlycontains the CTG leucine-specifying codon, while the DNA of yeasts andslime molds most commonly includes a TTA leucine-specifying codon. Inview of this hierarchy, it is generally believed that the likelihood ofobtaining high levels of expression of a leucine-rich polypeptide by anE. coli host will depend to some extent on the frequency of codon use.For example, it is likely that a gene rich in TTA codons will be poorlyexpressed in E. coli, whereas a CTG rich gene will probably be highlyexpressed in this host. Similarly, a preferred codon for expression of aleucine-rich polypeptide in yeast host cells would be TTA.

The implications of codon preference phenomena on recombinant DNAtechniques are manifest, and the phenomenon may serve to explain manyprior failures to achieve high expression levels of exogenous genes insuccessfully transformed host organisms—a less “preferred” codon may berepeatedly present in the inserted gene and the host cell machinery forexpression may not operate as efficiently. This phenomenon suggests thatsynthetic genes which have been designed to include a projected hostcell's preferred codons provide an optimal form of foreign geneticmaterial for practice of recombinant DNA techniques. Thus, one aspect ofthis invention is an HPV31 L1 gene that is codon-optimized forexpression in a yeast cell. In a preferred embodiment of this invention,it has been found that the use of alternative codons encoding the sameprotein sequence may remove the constraints on expression of HPV31 L1proteins by yeast cells.

In accordance with this invention, HPV31 L1 gene segments were convertedto sequences having identical translated sequences but with alternativecodon usage as described by Sharp and Cowe (Synonymous Codon Usage inSaccharomyces cerevisiae. Yeast 7: 657-678 (1991)), which is herebyincorporated by reference. The methodology generally consists ofidentifying codons in the wild-type sequence that are not commonlyassociated with highly expressed yeast genes and replacing them withoptimal codons for high expression in yeast cells. The new gene sequenceis then inspected for undesired sequences generated by these codonreplacements (e.g., “ATTTA” sequences, inadvertent creation of intronsplice recognition sites, unwanted restriction enzyme sites, etc.).Undesirable sequences are eliminated by substitution of the existingcodons with different codons coding for the same amino acid. Thesynthetic gene segments are then tested for improved expression.

The methods described above were used to create synthetic gene segmentsfor HPV31 L1, resulting in a gene comprising codons optimized for highlevel expression. While the above procedure provides a summary of ourmethodology for designing codon-optimized genes for use in HPV vaccines,it is understood by one skilled in the art that similar vaccine efficacyor increased expression of genes may be achieved by minor variations inthe procedure or by minor variations in the sequence.

Accordingly, the present invention relates to a synthetic polynucleotidecomprising a sequence of nucleotides encoding an HPV31 L1 protein, or abiologically active fragment or mutant form of an HPV31 L1 protein, thepolynucleotide sequence comprising codons optimized for expression in ayeast host. Said mutant forms of the HPV31 L1 protein include, but arenot limited to: conservative amino acid substitutions, amino-terminaltruncations, carboxy-terminal truncations, deletions, or additions. Anysuch biologically active fragment and/or mutant will encode either aprotein or protein fragment which at least substantially mimics theimmunological properties of the HPV31 L1 protein as set forth in SEQ IDNO:4. The synthetic polynucleotides of the present invention encode mRNAmolecules that express a functional HPV31 L1 protein so as to be usefulin the development of a therapeutic or prophylactic HPV vaccine.

One aspect of this invention is a codon-optimized nucleic acid moleculewhich encodes the HPV31 L1 protein as set forth in SEQ ID NO:4, saidnucleic acid molecule comprising a sequence of nucleotides as set forthin SEQ ID NO:2.

Another aspect of this invention is a codon-optimized nucleic acidmolecule which encodes the HPV31 L1 protein as set forth in SEQ ID NO:4,said nucleic acid molecule comprising a sequence of nucleotides as setforth in SEQ ID NO:3.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification.

The synthetic HPV31 DNA or fragments thereof constructed through themethods described herein may be recombinantly expressed by molecularcloning into an expression vector containing a suitable promoter andother appropriate transcription regulatory elements, and transferredinto prokaryotic or eukaryotic host cells to produce recombinant HPV31L1. Techniques for such manipulations are described in the art (Sambrooket al. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989); Current Protocols inMolecular Biology, Ausubel et al., Green Pub. Associates andWiley-Interscience, New York (1988); Yeast Genetics: A Laboratory CourseManual, Rose et al., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1990), which are hereby incorporated by reference in theirentirety).

Thus, the present invention further relates to a process for expressingan HPV31 L1 protein in a recombinant host cell, comprising: (a)introducing a vector comprising a nucleic acid encoding an HPV31 L1protein into a yeast host cell; wherein the nucleic acid molecule iscodon-optimized for optimal expression in the yeast host cell and; (b)culturing the yeast host cell under conditions which allow expression ofsaid HPV31 L1 protein.

The present invention also relates to a process for expressing an HPV31L1 protein in a recombinant host cell, comprising: (a) introducing avector comprising a nucleic acid encoding an HPV31 L1 protein into ayeast host cell; wherein the nucleic acid molecule is free from internaltranscription termination signals which are recognized by yeast and; (b)culturing the yeast host cell under conditions which allow expression ofsaid HPV31 L1 protein.

This invention further relates to a process for expressing an HPV31 L1protein in a recombinant host cell, comprising: (a) introducing a vectorcomprising a nucleic acid as set forth in SEQ ID NO:2 or SEQ ID NO:3into a yeast host cell; and, (b) culturing the host cell underconditions which allow expression of said HPV31 L1 protein.

The synthetic genes of the present invention can be assembled into anexpression cassette that comprises sequences designed to provideefficient expression of the HPV58 L1 protein in the host cell. Thecassette preferably contains the synthetic gene, with relatedtranscriptional and translations control sequences operatively linked toit, such as a promoter, and termination sequences. In a preferredembodiment, the promoter is the S. cerevisiae GAL1 promoter, althoughthose skilled in the art will recognize that any of a number of otherknown yeast promoters such as the GAL10, GAL7, ADH1, TDH3 or PGKpromoters, or other eukaryotic gene promoters may be used. A preferredtranscriptional terminator is the S. cerevisiae ADH1 terminator,although other known transcriptional terminators may also be used. Thecombination of GAL1 promoter—ADH1 terminator is particularly preferred.

Another aspect of this invention is an HPV31 virus-like particle (VLP),methods of producing HPV31 VLPs, and methods of using HPV31 VLPs. VLPscan self-assemble when L1, the major capsid protein of human and animalpapillomaviruses, is expressed in yeast, insect cells, mammalian cellsor bacteria (for review, see Schiller and Roden, in PapillomavirusReviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK:Leeds Medical Information, pp 101-12 (1996)). Morphologically indistinctHPV VLPs can also be produced by expressing a combination of the L1 andL2 capsid proteins. VLPs are composed of 72 pentamers of L1 in a T=7icosahedral structure (Baker et al., Biophys. J. 60(6): 1445-56 (1991)).

VLPs are morphologically similar to authentic virions and are capable ofinducing high titres of neutralizing antibodies upon administration intoan animal. Immunization of rabbits (Breitburd et al., J. Virol. 69(6):3959-63 (1995)) and dogs (Suzich et al., Proc. Natl. Acad. Sci. USA92(25): 11553-57 (1995) with VLPs was shown to both induce neutralizingantibodies and protect against experiment papillomavirus infection.However, because the VLPs do not contain the potentially oncogenic viralgenome and can self-assemble from a single gene, they present a safealternative to use of live virus in HPV vaccine development (for review,see Schiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000)).

Thus, the present invention relates to virus-like particles comprised ofrecombinant L1 protein or recombinant L1+L2 proteins of HPV31.

In a preferred embodiment of the invention, the HPV31 VLPs are producedin yeast. In a further preferred embodiment, the yeast is selected fromthe group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha,Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, andSchizosaccharomyces pombe.

Another aspect of this invention is an HPV31 VLP, which comprises anHPV31 L1 protein produced by a HPV31 L1 gene that is free from internaltranscription termination signals that are recognized by yeast.

Yet another aspect of this invention is an HPV31 VLP which comprises anHPV31 L1 protein produced by a codon-optimized HPV31 L1 gene. In apreferred embodiment of this aspect of the invention, thecodon-optimized HPV31 L1 gene consists essentially of a sequence ofnucleotides as set forth in SEQ ID NO:2 or SEQ ID NO:3.

Yet another aspect of this invention is a method of producing HPV31VLPs, comprising: (a) transforming yeast with a recombinant DNA moleculeencoding HPV31 L1 protein or HPV31 L1+L2 proteins; (b) cultivating thetransformed yeast under conditions that permit expression of therecombinant DNA molecule to produce the recombinant HPV31 protein; and(c) isolating the recombinant HPV31 protein to produce HPV31 VLPs.

In a preferred embodiment of this aspect of the invention, the yeast istransformed with a HPV31 L1 gene that is free from transcriptiontermination signals that are recognized by yeast. In another preferredembodiment, the yeast is transformed with a codon-optimized HPV31 L1gene to produce HPV31 VLPs. In a particularly preferred embodiment, thecodon-optimized HPV31 L1 gene consists essentially of a sequence ofnucleotides as set forth in SEQ ID NO:2 or SEQ ID NO:3.

This invention also provides a method for inducing an immune response inan animal comprising administering HPV31 virus-like particles to theanimal. In a preferred embodiment, the HPV31 VLPs are produced by a genethat is free from internal transcription termination sequences that arerecognized by yeast. In a further preferred embodiment, the HPV31 VLPsare produced by a codon-optimized gene.

Yet another aspect of this invention is a method of preventing ortreating HPV-associated cervical cancer comprising administering to amammal a vaccine comprising HPV31 VLPs. In a preferred embodiment ofthis aspect of the invention, the HPV31 VLPs are produced in yeast.

This invention also relates to a vaccine comprising HPV31 virus-likeparticles (VLPs).

In an alternative embodiment of this aspect of the invention, thevaccine further comprises VLPs of at least one additional HPV type. In apreferred embodiment, the at least one additional HPV type is selectedfrom the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV33, HPV35,HPV39, HPV45, HPV51, HPV52, HPV55, HPV56, HPV58, HPV59, and HPV68.

In a preferred embodiment of this aspect of the invention, the vaccinefurther comprises HPV16 VLPs.

In another preferred embodiment of the invention, the vaccine furthercomprises HPV16 VLPs and HPV18 VLPs.

In yet another preferred embodiment of the invention, the vaccinefurther comprises HPV6 VLPs, HPV11 VLPs, HPV16 VLPs and HPV18 VLPs.

This invention also relates to pharmaceutical compositions comprisingHPV 31 virus-like particles. Further, this invention relates topharmaceutical compositions comprising HPV31 VLPs and VLPs of at leastone additional HPV type. In a preferred embodiment, the at least oneadditional HPV type is selected from the group consisting of: HPV6,HPV11, HPV16, HPV18, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV55,HPV56, HPV58, HPV59, and HPV68.

Vaccine compositions of the present invention may be used alone atappropriate dosages defined by routine testing in order to obtainoptimal inhibition of HPV31 infection while minimizing any potentialtoxicity. In addition, co-administration or sequential administration ofother agents may be desirable.

The amount of virus-like particles to be introduced into a vaccinerecipient will depend on the immunogenicity of the expressed geneproduct. In general, an immunologically or prophylactically effectivedose of about 10 μg to 100 μg, and preferably about 20 μg to 60 μg ofVLPs is administered directly into muscle tissue. Subcutaneousinjection, intradermal introduction, impression though the skin, andother modes of administration such as intraperitoneal, intravenous, orinhalation delivery are also contemplated. It is also contemplated thatbooster vaccinations may be provided. Parenteral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration with adjuvants such as alum or Merck alum adjuvant,concurrently with or subsequent to parenteral introduction of thevaccine of this invention is also advantageous.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLE 1

Determination of a Representative HPV 31 L1 Sequence

The HPV 31 L1 wild-type sequence has been described previously(Goldsborough et al., Virology 171(1): 306-311 (1989); Genbank Accession# 304353). It is not uncommon, however, to find minor sequencevariations between DNAs obtained from clinical isolates. To isolate arepresentative HPV31 L1 wild-type sequence, DNA was isolated from threeclinical samples previously shown to contain HPV 31 DNA. HPV 31 L1sequences were amplified in a polymerase chain reaction (PCR) using TaqDNA polymerase and the following primers: HPV 31 L1 F 5′-CGT CGA CGT AAACGT GTA TCA TAT TTT TTT ACA G-3′ (SEQ ID NO:5) and HPV 31 L1 B 5′-CAGACA CAT GTA TTA CAT ACA CAA C-3′ (SEQ ID NO:6). The amplified productswere electrophoresed on agarose gels and visualized by ethidium bromidestaining. The ˜1500 bp L1 bands were excised and DNA purified using theQIA quick PCR purification kit (Qiagen, Hilden, Germany). The DNA wasthen ligated to the TA cloning vector, pCR-II (Invitrogen Corp.,Carlsbad, Calif.), E. coli transformed, and plated on LB agar withampicillin plus IPTG and X-gal for blue/white colony selection. Theplates were inverted and incubated for 16 hours at 37° C. White colonieswere cultured in LB medium with ampicillin, shaking at 37° C. for 16hours, and minipreps were performed to extract the plasmid DNA.

To demonstrate the presence of the L1 gene in the plasmid, restrictionendonuclease digestions were conducted and viewed by agarose gelelectrophoresis and ethidium bromide staining. DNA sequencing wasperformed on plasmids containing cloned L1 from each of the threeclinical isolates. DNA and translated amino acid sequences were comparedwith one another and the Genbank HPV 31 L1 sequences. Sequence analysisof the three clinical isolates revealed that no sequence was identicalto the Genbank sequence. The pCR-II-HPV 31L1/81 clone was chosen to bethe representative 31L1 sequence and is referred to herein as the “31 L1wild-type sequence” (SEQ ID NO:1, see FIG. 1). The sequence chosen as 31L1 wild-type contained one silent substitution at nucleotide 1266 and achange from a C to a G at nucleotide 1295, altering the encoded aminoacid from threonine to serine. The 31 L1 partial and total rebuilt genes(SEQ ID NOs: 2 and 3, respectively) also encode a serine at thislocation (see FIG. 1). In all cases, the amino acid sequences areidentical. Nucleotides were changed in the rebuilt constructs to encodeamino acids using yeast-preferred codon sequences and to eliminatepotential transcription termination signals (see EXAMPLE 2).

The 31 L1 wild-type sequence was amplified using the LS-101 5′-CTC AGATCT CAC AAA ACA AAA TGT CTC TGT GGC GGC CTA GC-3′ (SEQ ID NO:7) andLS-102 5′-GAC AGA TCT TAC TTT TTA GTT TTT TTA CGT ITT GCT GG-3′ (SEQ IDNO:8) primers to add BglII extensions. PCR was performed using Vent™ DNApolymerase. The PCR product was visualized by ethidium bromide stainingof an agarose gel. The ˜1500 bp band was excised and DNA purified usingthe QIAEX II gel extraction kit (Qiagen). The PCR product was thendigested with BglII at 37° C. for 2 hours and purified using the QIAquick PCR purification kit. The BglII digested 31 L1 PCR product wasligated to BamHI digested pGAL110 and DH5 E. coli were transformed.Colonies were screened by PCR for the HPV 31 L1 insert in the correctorientation. Sequence and orientation were confirmed by DNA sequencing.The selected clone was named pGAL110-HPV 31L1 #2.

Maxiprep DNA was then prepared and Saccharomyces cerevisiae were madecompetent and transformed. The yeast transformation was plated inLeu-sorbitol top-agar on Leu⁻ sorbitol plates and incubated inverted for3-5 days at 30° C. Colonies were picked and streaked for isolation onLeu⁻sorbitol plates. To induce L1 transcription and protein expression,isolated colonies were subsequently grown in 5 ml of 5×Leu⁻ Ade⁻sorbitol with 1.6% glucose and 4% galactose in rotating tube cultures at30° C.

EXAMPLE 2

Yeast Codon Optimization

Yeast-preferred codons have been described (Sharp and Cowe, Yeast 7:657-678 (1991)). Initially, the middle portion of HPV 31 L1,representing nucleotides 697-1249, was rebuilt utilizing yeast-preferredcodons. The strategy employed to rebuild was to design long overlappingsense and antisense oligomers that span the region to be rebuilt,substituting nucleotides with yeast-preferred codon sequences whilemaintaining the same amino acid sequence. These oligomers were used inplace of template DNA in the PCR reaction. Additional amplificationprimers were designed and used to amplify the rebuilt sequences fromtemplate oligomers with Pfu DNA polymerase (Stratagene, La Jolla,Calif.). The optimal conditions for amplification were section-specific;however, most employed a program resembling the following: an initialdenaturation step of 94° C. for 1 minute, followed by 15-25 cycles of95° C. for 30 sec denature, 55° C. for 30 sec anneal, 72° C. for 3.5minutes extension, followed by a 72° C. for 10 minute final extensionand 4° C. hold.

PCR products were examined by agarose gel electrophoresis. Bands of theappropriate size were excised and the DNA was gel purified. Theamplified fragments were then used as template to assemble the 552nucleotide rebuilt HPV 31 middle L1 fragment. PCR was then used toamplify the wild-type nucleotides 1-725 (5′end) and 1221-1515 (3′end). Afinal PCR using the 5′end, the 3′end, and the rebuilt middle wasperformed to generate full-length 31 L1 partial rebuild, referred toherein as the “31 L1 partial rebuild”.

The complete 31 L1 sequence was also rebuilt with yeast-preferredcodons. This construct is referred to herein as the “31 L1 totalrebuild”. Nine long overlapping oligomers were used to generateyeast-preferred codon nucleotide sequences from 1-753 and four longoverlapping oligomers were used to generate yeast-preferred codonnucleotide sequences from 1207-1515. After amplification and gelpurification, these fragments, along with the middle rebuilt sectiondescribed above (nucleotides 697-1249), were used together in a PCRreaction to generate the full length 31 L1 total rebuild sequence. Thispiece was generated with BamHI extensions. The gel purified rebuilt 31L1DNA was digested with BamHI, ligated to BamHI digested pGAL110expression vector and transformed into E. coli DH5 cells. Colonies werescreened by PCR for the HPV 31 L1 insert in the correct orientation.Sequence and orientation were confirmed by DNA sequencing.

Plasmid DNA was prepared. S. cerevisiae cells were made competent andtransformed. The yeast were plated in Leu⁻ sorbitol top-agar on Leu⁻sorbitol plates and incubated inverted for 3-5 days. Colonies werestreaked for isolation on Leu-sorbitol plates. Isolated colonies weresubsequently grown in 5 ml of 5×Leu- Ade-sorbitol with 1.6% glucose and4% galactose in rotating tube cultures at 30° C. to induce L1transcription and protein expression. After 48-72 hours, culture volumeequivalent to an OD600=10 was pelleted, supernate removed and thepellets frozen and stored −70° C.

EXAMPLE 3

RNA Preparation

Cell pellets of transformed yeast, which were induced to express HPV 31L1 by galactose induction, were thawed on ice and suspended in 1 ml ofcold DEPC-treated water. Cells were pelleted by centrifugation and theresulting supernatant was removed. The cell pellet was then resuspendedin 400 μl TES (10 mM Tris pH7.0, 10 mM EDTA and 0.5% SDS). An equalvolume of AE buffer-saturated phenol (50 mM NaOAc and 10 mM EDTA) wasadded. The tube was vortexed for 10 seconds and heated to 65° C. for 50minutes with mixing every 10 minutes. The tube was then placed on icefor 5 minutes, followed by centrifugation at 4° C. for 5 minutes. Thesupernatant was collected and transferred to a sterile tube. Anadditional 400 μl of phenol was added, the tube vortexed, placed on icefor 5 minutes and centrifuged. The supernatant was transferred to asterile tube and 400 μl of chloroform added, mixed and centrifuged. Thesupernatant was again collected and transferred to a sterile tube and 40μl 3 M Na Acetate pH 5.2 added in addition to 1 ml 100% EtOH. The tubewas placed on dry ice for one hour, after which it was centrifuged athigh speed to pellet the RNA. The RNA was washed one time with 70% EtOHand air-dried. The RNA was then suspended in 100 μl DEPC-treated waterand heated to 65° C. for 5 minutes to dissolve. Spectrophotometry wasperformed to determine the concentration of RNA in the sample using theassumption that an A260 reading of 1=40 μg/ml RNA when the A260/280 is1.7-2.0.

EXAMPLE 4

Northern Blot Analysis

Initial analysis of yeast expressing 31 L1 wild-type suggested that theexpression yield of HPV 31 L1 protein was considerably less than wasexpected. To determine if the low expression was occurring due to aproblem at the transcription level versus the translation level,Northern blot analysis of the HPV 31 L1 transcript was performed.Northern blots were made from gels in which RNA from yeast expressingHPV16 L1 was run with RNA from yeast expressing HPV31 L1 on the same gelto compare transcript sizes.

A 1.2% agarose formaldehyde gel was cast. Ten micrograms of RNA wascombined with denaturing buffer (final concentrations: 6% formaldehyde,45% formamide and 0.9×MOPS) and heated to 55° C. for 15 minutes. Aone-tenth volume of gel loading buffer was added and the sample loadedonto the gel. Electrophoresis was performed at 65 volts in 1×MOPS bufferfor ˜5 hours. The gel was washed for 15 minutes in sterile waterfollowed by two five minute washes in 10×SSC. The RNA was transferred toa Hybond-N+ nylon membrane (Amersham Biosciences, Piscataway, N.J.) bycapillary action over 16 hours in 10×SSC. The RNA was then fixed to thenylon membrane by cross-linking using the Amersham cross-linker set for700 units of energy. After fixing, the nylon membrane was allowed to airdry. The membrane was placed in 30 ml Zetaprobe buffer at 55° C. for 2hours after which 32P-labeled probes were added and incubated for 16hours at 53-65° C. The membrane was then washed 3 times in 5×SSC at roomtemperature for 20 minutes, followed by 2 times in 0.4×SSC for 20minutes at room temperature and once at 60° C. for 10 minutes. Probe DNAwas generated by PCR using HPV 31 L1 sequence specific sense andantisense primers. The amplified DNA was labeled by treatment withpolynucleotide kinase (PNK) and γ-32P ATP at 37° C. for 1 hour. The blotwas wrapped in saran wrap and exposed to x-ray film for 16 hours. Uponfilm development, probe-hybridized RNA was detected as a black band onthe autoradiograph.

Analysis of the Northern blot described above revealed that the majorityof the full-length HPV 31 L1 wild-type transcripts were considerablysmaller than full length (see FIG. 4). However, the 31 L1 partialrebuild was designed not only to insert yeast-preferred codons in themiddle of the gene, but also to eliminate any potential sequencesresembling yeast transcription termination sites. Northern blot analysisclearly showed that upon rebuilding, the length of the 31 L1 genetranscript had significantly increased to a size corresponding with thatof the full-length HPV 16 L1 transcript (not shown). Thus, prematuretranscription termination is likely to have accounted for a significantportion of the low expression yield from the 31 L1 wild-type construct.

EXAMPLE 5

HPV 31 L1 Protein Expression

Frozen yeast cell pellets of galactose induced cultures equivalent toOD600=10 were thawed on ice and suspended in 300 μl of PC buffer (100 mMNa2HPO4 and 0.5 M NaCl, pH 7.0) with 2 mM PMSF. Acid-washed 0.5 mm glassbeads were added, ˜0.5 g/tube. The tubes were vortexed for 15 minutes at4° C. 7.5 μl of 20% TritonX100 was added and vortex repeated for 5minutes at 4° C. The transferred to a sterile microcentrifuge tube andstored at −70° C.

EXAMPLE 6

Western Blot Analysis

Total yeast protein extract from twenty to forty isolated yeast coloniesfor each HPV 31 L1 construct were analyzed by Western blot to confirmexpression of HPV 31 L1 protein after galactose induction.

Ten micrograms of total yeast protein extract was combined with SDS-PAGEloading buffer and heated to 95° C. for 10 minutes. The proteins wereloaded onto an 8% SDS-PAGE gel and electrophoresed in Tris-Glycinebuffer. After protein separation, the proteins were Western transferredfrom the gel to nitrocellulose and the blot was blocked in 10% non-fatdry milk in TTBS (Tris buffered saline with Tween-20) for 16 hours. Theblot was washed three times in TTBS. Goat anti-trpE-HPV 16 L1 serum, apolyclonal serum that cross-reacts with HPV 31 L1, was applied at a1:1000 dilution in TTBS for 1 hr at room temperature. The blot waswashed three times in TTBS and anti-goat-HRP conjugated antibody wasapplied at a 1:2500 dilution in TTBS for 1 hr. The blot was again washedthree times and ECL™ detection reagent was applied (AmershamBiosciences, Piscataway, N.J.). Autoradiography was then performed.Proteins recognized by the antiserum were visualized by the detectionreagent as dark bands on the autoradiograph.

In all cases, the HPV 31 L1 protein was detected as a distinct band onthe autoradiograph corresponding to approximately 55 kD (data notshown). The HPV 16 L1 protein was included as a positive control on thegels.

EXAMPLE 7

Radioimmunoassay (RIA)

The yeast cells expressing HPV 31 L1 were grown by a variety of methods,including rotating tube cultures, shake flasks and fermenters. The yeastwere lysed and protein extracts made to determine the amount of HPV 31L1 virus-like particles (VLPs) produced per milligram of total protein.To demonstrate HPV 31 L1 VLP expression, a portion of each total yeastprotein extract was analyzed by capture radioimmunoassay (RIA).

The RIA was performed using a detection monoclonal antibody, H31.A6,that is HPV type 31-specific and VLP conformational-specific. H31.A6 isspecific for HPV type 31 L1 as it is found to bind intact HPV 31 L1 VLPsand does not recognize denatured HPV 31 VLPs. This mAb can besubsequently detected by a goat anti-mouse antibody radiolabeled withI125. Therefore, the counts per minute (cpm) values correspond torelative levels of HPV31 L1 VLP expression.

Polystyrene beads were coated with a goat anti-trpE-HPV31 L1 polyclonalserum diluted 1:1000 in PBS overnight. The beads were then washed with 5volumes of sterile distilled water and air-dried. The antigen, totalyeast protein extract from isolated yeast colonies, was then loaded ontothe beads by dilution in PBS with 1% BSA, 0.1% Tween-20 and 0.1% NaAzide and incubated with rotation for one hour. After washing, the beadswere distributed one per well in a 20-well polystyrene plate andincubated with H31.A6 mAb diluted 1:50,000 for 17-24 hours at roomtemperature. The beads were washed and I125 labeled goat anti-mouse IgGwas added at an activity range of 23000-27000 cpm per 10 μl. After 2hours, the beads were washed and radioactive counts were recorded incpm/ml. Background counts from blank wells were subtracted from thetotal cpm/ml, giving the RIA minus background value.

Two experiments were performed: in experiment 1, protein extracts from31 L1 wild-type and 31 L1 partial rebuild were compared and inexperiment 2, protein extracts from 31 L1 partial rebuild and 31 L1total rebuild were compared (see FIG. 5). Results indicate that 31 L1partial rebuild VLP expression is 6.9 fold greater than 31 L1 wild-type.The 31 L1 total rebuild has a 1.7 fold increased expression over the 31L1 partial rebuild. Therefore, the 31 L1 expression levels wereincreased >7 fold by introducing yeast-preferred codon sequences andeliminating potential transcription termination signals.

EXAMPLE 8

Transmission Electron Microscopy

To demonstrate that the HPV 31 L1 protein was in fact self-assembling toform pentameric-L1 capsomers, which in turn self-assemble intovirus-like particles, a partially purified 31 L1 total rebuild proteinextract was subject to transmission electron microscopy (TEM). Yeastwere grown under small scale fermentation and pelleted. The pellets weresubjected to purification treatments. Pellet and clarified yeastextracts were analyzed by immunoblot to demonstrate L1 proteinexpression and retention through the purification procedure. Clarifiedyeast extracts were then subjected to centrifugation over a 45%-sucrosecushion and the resulting pellet suspended in buffer for TEM analysis(see FIG. 6). Results indicated that the diameter of the sphericalparticles in this crude sample ranged from between 30 and 60 nm withsome particles displaying a regular array of capsomers.

1. A nucleic acid molecule comprising a sequence of nucleotides thatencodes an HPV31 L1 protein as set forth in SEQ ID NO:4, the nucleicacid sequence being codon-optimized for high level expression in a yeastcell.
 2. A vector comprising the nucleic acid molecule of claim
 1. 3. Ahost cell comprising the vector of claim
 2. 4. The host cell of claim 3,wherein the host cell is selected from the group consisting of:Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris,Kluyvermycesfragilis, Kluyveromyces lactis, and Schizosaccharomycespombe.
 5. The host cell of claim 4, wherein the host cell isSaccharomyces cereviszae.
 6. The nucleic acid molecule of claim 1,wherein the sequence of nucleotides comprises a sequence of nucleotidesas set forth in SEQ ID NO:2 or SEQ ID NO:3.
 7. A vector comprising thenucleic acid molecule of claim
 6. 8. A host cell comprising the vectorof claim 7.